1. Field of the Invention
The present invention relates to heregulin variants, nucleic acid molecules encoding such variants, and related vectors, host cells, pharmaceutical compositions, and methods. In particular, the invention relates to amino acid substitution variants of human heregulin-xcex21 having an enhanced affinity for the ErbB-3 and ErbB-4 receptors.
2. Description of the Related Art
Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases are enzymes that catalyze this process. Receptor protein tyrosine kinases are believed to direct cellular growth via ligand-stimulated tyrosine phosphorylation of intracellular proteins. Growth factor receptor protein tyrosine kinases of the class I subfamily include the 170 kilodalton (kDa) epidermal growth factor receptor (EGFR) encoded by the erbB1 gene. erbB1 has been causally implicated in human malignancy. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung, and stomach.
The second member of the class I subfamily, p185neu (also called the ErbB-2 receptor or p185HER2), was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The neu (erbB2 or HER2) gene encodes a 185 kDa receptor protein tyrosine kinase.
Amplification and/or overexpression of the human erbB2 gene correlates with a poor prognosis in breast and ovarian cancers. Slamon et al., Science 235:177-82 (1987); Slamon et al., Science 244:707-12 (1989). Overexpression of erbB2 has been correlated with other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder. Accordingly, in U.S. Pat. No. 4,968,603, Slamon et al. describe and claim various diagnostic assays for determining erbB2 gene amplification or expression in tumor cells. Slamon et al. discovered that the presence of multiple copies of the erbB2 oncogene in tumor cells indicates that the disease is more likely to spread beyond the primary tumor site, and that the disease may therefore require more aggressive treatment than might otherwise be indicated by other diagnostic factors. Slamon et al. conclude that the erbB2 gene amplification test, together with the determination of lymph node status, provides greatly improved prognostic utility.
A further related gene, called erbB3 (or HER3), which encodes the ErbB-3 receptor (p180HER3) has also been described. See U.S. Pat. No. 5,183,884; Kraus et al., PNAS USA 86:9193-97 (1989); EP Patent Application No. 444,961A1; Kraus et al., PNAS USA 90:2900-04 (1993). Kraus et al. (1989) discovered that markedly elevated levels of erbB3 mRNA were present in certain human mammary tumor cell lines indicating that erbB3, like erbB1 and erbB2, may play a role in human malignancies. Also, Kraus et al. (1993) showed that EGF-dependent activation of the ErbB-3 catalytic domain of a chimeric EGFR/ErbB-3 receptor resulted in a proliferative response in transfected NIH-3T3 cells. Furthermore, these researchers demonstrated that some human mammary tumor cell lines display a significant elevation of steady-state ErbB-3 receptor tyrosine phosphorylation, further implicating this receptor in human malignancies. The role of erbB3 in cancer has been explored by others, and this gene has been found to be overexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-21 [1992]), gastrointestinal (Poller et al., J. Pathol. 168:275-80 [1992]; Rajkumer et al., J. Pathol. 170:271-78 [1993]; Sanidas et al., Int. J. Cancer 54:935-40 [1993]), and pancreatic cancers (Lemoine et al., J. Pathol. 168:269-73 [1992], and Friess et al., Clinical Cancer Research 1:1413-20 [1995]).
The class I subfamily of growth factor receptor protein tyrosine kinases has been further extended to include the ErbB-4 (HER4) receptor, which is the product of the erbB4 (HER4) gene. See EP Patent Application No. 599,274; Plowman et al., PNAS USA 90:1746-50 (1993); and Plowman et al., Nature 366:473-75 (1993). Plowman et al. found that increased erbB4 expression closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Diagnostic methods for detection of human neoplastic conditions (especially breast cancers) that evaluate erbB4 expression are described in EP Patent Application No. 599,274.
The quest for the activator of the erbB2 oncogene has lead to the discovery of a family of heregulin polypeptides. In humans, the heregulin polypeptides characterized thus far are derived from alternate splicing of a single gene which was mapped to the short arm of chromosome 8 by Lee and Wood, Genomics 16:790-91 (1993).
Holmes et al. isolated and cloned a family of polypeptide activators for the ErbB-2 receptor which they called heregulin-xcex1 (HRG-xcex1), heregulin-xcex21 (HRG-xcex21), heregulin-xcex22 (HRG-xcex22), and heregulin-xcex23 (HRG-xcex23). See Holmes et al., Science 256:1205-10 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060. These researchers demonstrated the ability of the purified heregulin polypeptides to activate tyrosine phosphorylation of the ErbB-2 receptor in MCF7 breast tumor cells. Furthermore, the mitogenic activity of the heregulin polypeptides on SK-BR-3 cells (which express high levels of the ErbB-2 receptor) was also demonstrated.
Heregulins are large multi-domain proteins that are typically expressed as xe2x80x9cpro-heregulins.xe2x80x9d Pro-heregulins have been shown to undergo proteolytic processing to a mature soluble form (usually of about 44-45 kDa). Processing has been shown to occur intracellularly or at the cell surface. Domains in the soluble form include (in order from the N- to the C-terminus) an immunoglobulin homology (Ig-like) domain, a spacer region rich in glycosylation sites, and a domain similar to a domain found in EGF that is sufficient for ErbB receptor binding and activation. See Barbacci, et al., J. Biol. Chem. 270:9585-89 (1995).
The heregulin EGF-like domains are characterized by substantial structural similarities to (Jacobsen et al., Biochemistry 35:3402-17 [1996]), and limited sequence homology with, EGF residues 1-48 (Holmes, et al., supra). Functional similarities between the heregulin EGF-like domains and EGF have been established by data showing that blocks of EGF sequence substituted into heregulin-xcex21 do not impair binding to cells co-expressing ErbB-3 and ErbB-2. Barbacci et al., supra.
While heregulins are substantially identical in the first 213 amino acid residues, they are classified into two major types, xcex1 and xcex2, based on two EGF-like domains that differ in their C-terminal portions. For example, the heregulin-xcex1 EGF-like domain differs from that of the xcex21-isoform by nine substitutions near the C-terminus. The xcex2-isoform has been reported to bind ErbB receptors with approximately eight to 10-fold higher affinity than the xcex1-isoform. Wen et al., Mol. Cell. Biol. 14:1909-19 (1994).
The solution structure of the heregulin-xcex1 EGF domain has recently been determined at high resolution by NMR. Jacobsen et al., supra; Nagata et al., EMBO J. 13, 3517-3523 (1994). The salient features of this domain include (1) an N-terminal subdomain containing a central three-stranded xcex2-sheet with an intermittent helix and (2) a smaller C-terminal subdomain that contains a short stretch of xcex2-sheet. The EGF domain is stabilized by three disulfide bonds, two in the N-terminal subdomain and one in the C-terminal subdomain. The pairing of the six corresponding cysteine residues is conserved in EGF-like domains from all heregulins and from EGF.
The 44 kDa neu differentiation factor (NDF), which is the rat equivalent of human HRG, was first described by Peles et al., Cell, 69:205-16 (1992), and Wen et al., Cell, 69:559-72 (1992). Like the human heregulin polypeptides, NDF has an Ig-like domain followed by an EGF-like domain and lacks a N-terminal signal peptide. Subsequently, Wen et al. carried out xe2x80x9cexhaustive cloningxe2x80x9d to extend the family of NDFs. Wen et al., Mol. Cell. Biol., 14:1909-19 (1994). This work revealed six distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmes et al., the NDFs were classified as either xcex1 or xcex2 polypeptides based on the sequences of the EGF-like domains. Isoforms 1 to 4 are characterized on the basis of a variable region between the EGF-like domain and transmembrane domain. Also, isoforms a, b and c are defined based on variable-length cytoplasmic domains. These researchers conclude that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions. See also EP 505 148; WO 93/22424; and WO 94/28133 (discussing NDF).
Falls et al., Cell 72:801-815 (1993) describe another member of the heregulin family which they call xe2x80x9cacetylcholine receptor inducing activity (ARIA) polypeptide.xe2x80x9d The chicken-derived ARIA polypeptide stimulates synthesis of muscle acetylcholine receptors. See WO 94/08007. ARIA is a xcex2-type heregulin and lacks the entire spacer region between the Ig-like domain and EGF-like domain of HRG-xcex1 and HRGxcex21-xcex23.
Marchionni et al., Nature 362:312-318 (1993) identified several bovine-derived proteins that they call xe2x80x9cglial growth factors (GGFs).xe2x80x9d These GGFs share the Ig-like domain and EGF-like domain with the other heregulin proteins described above, but also have an amino-terminal kringle domain. GGFs generally do not have the complete spacer region between the Ig-like domain and EGF-like domain. Only one of the GGFs, GGFII, has an N-terminal signal peptide. See also WO 92/18627; WO 94/00140; WO 94/04560; WO 94/26298; WO 95/32724 (describing GGFs and uses thereof).
Ho et al. describe another member of the heregulin family called xe2x80x9csensory and motor neuron-derived factor (SMDF).xe2x80x9d Ho et al., J. Biol. Chem. 270:14523-32 (1995). This protein has an EGF-like domain characteristic of all other heregulin polypeptides but a distinct N-terminal domain. In addition, SMDF lacks both the Ig-like domain and the spacer region found in other heregulin polypeptides. Another feature of SMDF is the presence of two stretches of hydrophobic amino acids near the N-terminus.
While the heregulin polypeptides were first identified based on their ability to activate the ErbB-2 receptor (see Holmes et al., supra), it has been discovered that certain ovarian cells expressing neu (erbB2) and neu-transfected fibroblasts did not bind or crosslink to NDF, nor did they undergo tyrosine phosphorylation in response to NDF. Peles et al., EMBO J. 12:961-71 (1993). This finding indicated that another cellular component was necessary for conferring full heregulin responsiveness. .
Carraway et al. subsequently demonstrated that 125I-rHRG-xcex21 177-244 bound to NIH-3T3 fibroblasts stably transfected with bovine erbB3 but not to non-transfected parental cells. These researchers also expressed bovine ErbB-3 receptor in insect cells and showed that HRG-xcex21 177-244 bound to a preparation of ErbB-3 receptor solubilized from these cells. They concluded that ErbB-3 is a receptor for heregulin and mediates phosphorylation of intrinsic tyrosine residues as well as phosphorylation of ErbB-2 receptor in cells that express both receptors. Carraway et al., J. Biol. Chem. 269:14303-06 (1994). Sliwkowski et al. found that cells transfected with erbB3 alone show low affinities for heregulin, whereas cells transfected with both erbB2 and erbB3 show higher affinities. Sliwkowski et al., J. Biol. Chem. 269:14661-65 (1994).
Plowman and his colleagues have similarly studied ErbB-4/ErbB-2 receptor activation. They expressed the ErbB2 receptor alone, the ErbB4 receptor alone, or the two receptors together in human T lymphocytes and demonstrated that heregulin is capable of stimulating tyrosine phosphorylation of ErbB-4, but could only stimulate ErbB-2 phosphorylation in cells expressing both receptors. Plowman et al., Nature 336:473-75 (1993).
These observations are consistent with the xe2x80x9creceptor cross-talkingxe2x80x9d concept described previously by Kokai et al., Cell 58: 287-92 (1989), Stern et al., EMBO J. 7:995-1001 (1988), and King et al., 4:13-18 (1989). These researchers found that binding of EGF to the EGFR resulted in activation of the EGFR kinase domain and cross-phosphorylation of the ErbB-2 receptor. This is believed to be a result of ligand-induced receptor heterodimerization and the concomitant cross-phosphorylation of the receptors within the heterodimer. Wada et al., Cell 61:1339-47 (1990).
Thus, the ErbB receptors are believed to be activated by ligand-induced receptor dimerization. Specifically, heregulins can bind separately to ErbB-3 and ErbB-4 receptors, but not to the ErbB-2 receptor. However, ErbB-2 is required for signalling, and heterodimers containing ErbB-2 in combination with ErbB-3 or ErbB-4 bind heregulins with higher affinity than homodimers containing ErbB-3 or ErbB-4. Plowman et al., Nature 366:473-75 (1993); Sliwkowski et al., J. Biol. Chem. 269:14661-65 (1994).
The biological activities of heregulins have been investigated by several groups. For example, Holmes et al. (supra) found that heregulin exerts a mitogenic effect on mammary cell lines (such as SK-BR-3 and MCF-7). Lewis et al. reported that heregulin-xcex21 stimulated proliferation and enhanced colony formation in soft agar in a number of human breast and ovarian tumor cell lines. Lewis et al., Cancer Research 56:1457-65 (1996). These researchers also showed that ErbB-2 is a critical mediator of heregulin responsiveness.
Pinkas-Kramarski et al. found that NDF (rat heregulin) is expressed in neurons and glial cells in embryonic and adult rat brain and primary cultures of rat brain cells, and suggested that NDF may act as a survival and maturation factor for astrocytes. Pinkas-Kramarski et al., PNAS USA 91:9387-91 (1994). Danilenko et al. reported that the interaction of NDF and the ErbB-2 receptor is important in directing epidermal migration and differentiation during wound repair. Danilenko et al., Abstract 3101, FASEB 8(4-5):A535 (1994).
Meyer and Birchmeier analyzed expression of mouse heregulin during embryogenesis and in the perinatal animal using in situ hybridization and RNase protection experiments. Meyer and Birchmeier, PNAS USA 91:1064-68 (1994). These authors conclude, based on expression of this molecule, that heregulin plays a role in vivo as a mesenchymal and neuronal factor. Their findings also indicated that heregulin functions in the development of epithelia.
Falls et al. (supra) found that chicken ARIA plays a role in myotube differentiation, namely affecting the synthesis and concentration of neurotransmitter receptors in the postsynaptic muscle cells of motor neurons. Corfas and Fischbach demonstrated that ARIA also increases the number of sodium channels in chick muscle. Corfas and Fischbach, J. Neuroscience 13:2118-25 (1993).
Bovine GGFs have been reported to be mitogenic for Schwann cells. See, e.g., Brockes et al., J. Biol. Chem. 255:8374-77 (1980); Lemke and Brockes, J. Neurosci. 4:75-83 (1984); Brockes et al., J. Neuroscience 4:75-83 (1984); Brockes et al., Ann. Neurol. 20:317-22 (1986); Brockes, Methods in Enzym. 147:217-225 (1987); Marchionni et al., supra. Schwann cells provide myelin sheathing around the axons of myelinated neurons and thus play an important role in the development, function and regeneration of peripheral nerves. The implications of this role from a therapeutic standpoint have been addressed by Levi et al., J. Neuroscience 14:1309-19 (1994). Levi et al. discussed the potential for construction of a cellular prosthesis including Schwann cells that could be transplanted into areas of damaged spinal cord. Methods for culturing Schwann cells ex vivo have been described. See WO 94/00140; Li et al., J. Neuroscience 16:2012-19 (1996).
GGFII has been shown to be mitogenic for subconfluent quiescent human myoblasts, and differentiation of clonal human myoblasts in the continuous presence of GGFII results in greater numbers of myotubes after six days of differentiation. Sklar et al., J. Cell Biochem., Abst. W462, 18D, 540 (1994); see also WO 94/26298.
The relationship between the structure and function of new proteins can be investigated using any of a variety of available mutational analysis techniques. Examples of such techniques include alanine scanning mutagenesis and phagemid display. Alanine scanning can be used to identify active residues (i.e., residues that have a significant effect on protein function) in a protein or protein domain. For example, Cunningham and Wells used alanine scanning to identify residues in human growth hormone that were important for binding its receptor. Cunningham and Wells, Science 244:1081-85 (1989). In alanine scanning, a gene encoding the protein or domain to be scanned is inserted into an expression vector, and mutagenesis is carried out to generate a series of vectors that encode proteins or domains in which sequential residues are converted to alanine. The encoded proteins or domain are expressed from these vectors, and the activities of the alanine-substituted variants are then tested to identify those with altered activity. An alteration in activity indicates that the residue at the alanine-oubatituted position is an active residue.
Phagemid display was developed to allow the screening of a large number of variant polypeptides for a particular binding activity. Smith and Parmley demonstrated that foreign peptides can be xe2x80x9cdisplayedxe2x80x9d efficiently on the surface of filamentous phage by inserting short gene fragments into gene III of the fd phage. Smith, Science 228:1315-17 (1985); Parmley and Smith, Gene 73:305-18 (1985). The gene III coat protein is present in about five copies at one end of the phage particle. The modified phage were termed xe2x80x9cfusion phagexe2x80x9d because they displayed the foreign peptides fused to the gene III coat protein. As each fusion phage particle displayed approximately five copies of the fusion protein, this mode of phage display was termed xe2x80x9cpolyvalent display.xe2x80x9d
Scott et al. and Cwirla et al. showed that fusion phage libraries could be screened by sequential affinity selections known as xe2x80x9cpanning.xe2x80x9d Scott et al., Science 249:386-90 (1990); Cwirla et al., PNAS USA 87:6378-82 (1990). However, early efforts to select high affinity fusion phage failed, presumably due to the polyvalence of the phage particles. This problem was solved with the development of a xe2x80x9cmonovalentxe2x80x9d phage display system in which the fusion protein is expressed at a low level from a phagemid and a helper phage provides a large excess of wild-type coat protein. Bass et al., Proteins 8:309-14 (1990); Lowman et al., Biochem. 30:10832-38 (1991). Monovalent phage display can be used to generate and screen a large number of variant polypeptides to isolate those that bind with high affinity to a target of interest.
The present invention provides a heregulin variant having an amino acid sequence not found in nature and the ability to bind an ErbB receptor. In one embodiment, the variant has an amino acid substitution at a selected residue corresponding to a residue of 645-amino acid native human heregulin-xcex21 selected from the group consisting of:
S177, H178, L179, V180, K181, E184, E186,
K187, T188, V191, N192, G193, G194, E195,
M198, V199, K200, D201, N204, P205, S206,
R207, Y208, L209, K211, P213, N214, E215,
T217, G218, D219, Q222, N223, Y224, S228, and F229.
In a variation of this embodiment, the amino acid substitution is not a replacement of the selected residue with an epidermal growth factor (EGF) residue corresponding to the selected residue.
The heregulin variant can be a variant of any member of the heregulin family from any species. In one embodiment, the heregulin variant is a variant of a human heregulin, such as, for example, human heregulin-xcex21. The invention provides a human heregulin-xcex21 variant including an amino acid substitution selected from the group consisting of:
S177W; H178S, E, R, or A; V18OQ, I, or E;
K181P or A; A183G; E184V, W, K, R, G, or N;
K16SE, S, Q, or G; E186R; K187E or A; T188Q;
E195Q; F197Y; M198R or K; K20OR; D201T or I;
P20ST or Y; S206K, H, G, P, or R; R207Y;
Y208R or L; L209M or G; K211R; P213S, T, N, or K; N214L, K, S, or E; F216M; N223H or W; and M226I.
In a variation of this embodiment, the heregulin variant includes sets of amino acid substitutions selected from this group. Some heregulin variants of the invention having sets of amino acid substitutions exhibit at least a 50-fold increase in ErbB-3 receptor affinity, which is also accompanied by an increase in ErbB-4 receptor affinity.
The invention also includes a heregulin variant that has a greater specificity for the ErbB-4 receptor, relative to the ErbB-3 receptor, than the heregulin from which the heregulin variant is derived. In one embodiment, this heregulin variant has an amino acid substitution at a selected residue corresponding to a residue of 645-amino acid native human heregulin-xcex21 selected from the group consisting of H178, L179, and R207.
In another embodiment, a heregulin variant having a greater specificity for the ErbB-4 receptor, relative to the ErbB-3 receptor, has a deletion of amino acid residues corresponding to residues S228 to K231 of 645-amino acid native human heregulin-xcex21 and a substitution of a single methionine for the deleted residues.
In addition to including one or more of the mutations disclosed herein, the heregulin variant can have one or more other modifications, such as an amino acid substitution, an insertion of at least one amino acid, a deletion of at least one amino acid, or a chemical modification. For example, the invention provides a heregulin variant that is a fragment. In a variation of this embodiment, the fragment includes residues corresponding to a portion of human heregulin-xcex21 extending from about residue 175 to about residue 230 (i.e., the EGF-like domain). In a further variation of this embodiment, the fragment includes residues corresponding to a portion of human heregulin-xcex21 extending from about residue 175 to about residue 245.
One aspect of the invention is a method for producing such a modified heregulin variant. The modification(s) is selected so that the modified heregulin variant retains the ability to an ErbB receptor.
In addition to a heregulin variant, the invention provides a related nucleic acid molecule, vector, and host cell. The invention also provides a method of producing a heregulin variant in which a host cell containing an expression vector capable of expressing the heregulin variant is cultured under conditions that allow expression of the heregulin variant, and the heregulin variant then recovered from the culture.
Other aspects of the invention relate to various uses of a heregulin variant. For example, the invention provides a method for activating an ErbB receptor in which the heregulin variant is contacted with a cell that expresses an ErbB receptor. The heregulin variant can be contacted with cells in culture, for example, to promote ex vivo survival, proliferation, or differentiation of cells, such as glial, Schwann, or muscle cells.
Alternatively, the heregulin variant can be combined with a pharmaceutically acceptable carrier and used to treat one of a wide range of cancers as well as diseases and disorders affecting the nervous system, musculature, and epithelia. Thus, the present invention provides a pharmaceutical composition and a treatment method.
The invention also includes a method of determining whether a sample contains an ErbB receptor that binds a heregulin. In particular, a heregulin variant is contacted with a sample, and specific binding between the heregulin variant and a component of the sample is determined as an indication of the presence and/or amount of ErbB receptor(s) present in the sample.